1. Technical Field
The present invention relates to production of protein or polypeptide drugs using recombinant DNA techniques, and specifically, relates to a method of producing lysostaphin by secretory expression in Escherichia coli. 
2. Background Art
Lysostaphin was first found by Schindler and Schuhard (U.S. Pat. No. 3,278,378; 1966) in the culture of Staphylococcus simulans in 1964, which has a molecular weight of 27 kDa, and consists of 246 amino acids. The lysostaphin, which is an endopeptidase, functions through lysing the pentaglycine cross-links in Staphylococcus cell wall peptidoglycan, thereby disrupting the integrity of the cell wall and lysing the bacterial cell body. The lysostaphin gene has been expressed in Escherichia coli, Bacillus subtitlis and Bacillus sphaericus etc. utilizing molecular cloning method in China and abroad since 1980s. Because of its unique mechanism of killing bacteria, lysostaphin is able to lyse the cell wall of Staphylococcus quickly at a low concentration, hence it has an immediate action and a robust activity against the bacteria. In addition, a resistant bacterial strain is rarely induced by lysostaphin, and lysostaphin has a specific antibacterial spectrum. Recombinant lysostaphin in high purity can be commercially manufactured in China and abroad nowadays, and it has been widely used in studies of bacteriology, disinfection, and enzymology, and in clinically anti-bacterial therapy etc.
Since Staphylococus simulans belongs to a pathogen and produces only limited amount of lysostaphin, and for sake of the recent development of molecular biology technology, Recsei et al from the U.S.A. utilized the molecular cloning method to express lysostaphin gene in Escherichia coli JM 105, Bacillus subtilis and Bacillus sphaericus in 1987. The amount of lysostaphin expressed in Escherichia coli JM 105 is 3 ug/ml, of which 65% exists in the culture supernatant, 15% in the cell periplasm, and 20% in the cytoplasm. A patent was granted in 1990, with the patent No. 4931390, which discloses inserting a 1.5 kb lysostaphin-encoding DNA fragment into vector pUC8, pBC16, pBD64 or pSPV1 to form the recombinant plasmid pRG5, pJP1, pDF8 or pRP1, respectively. pRG5 is used to transform Escherichia coli JM105. pJP1, pDF8, and pRP1 are used to transform various Bacillus (Bacillus subtilis BD170, Bacillus sphaericus 00), the lysostaphin produced thereby is identified by immunological techniques and electrophoresis, then compared with lysostaphin produced by S. simulans. The amount of lysostaphin (150 mg/L) produced from Bacillus sphaericus transformants is 5 times of that produced by S. simulans. This referenced patent also provides a 1.5 kb DNA sequence, which encodes a precursor of prolysostaphin with 389 amino acids, and this precursor is processed to be mature lysostaphin after translation. Recently it has been found that lysostaphin has heterogeneous N-terminal, most of them have two amino acids shorter than that of wild-type lysostaphin.
WALTER P. HAMMES et al from Germany expressed the lysostaphin gene with the precursor deleted in Meat Lactobacilli (a lactobacillus) in 1996. The aim for deleting the precursor is to acquire stable expression in Meat Lactobacilli, please see patent No.: EP0759473; 1997, but this method remains to be intracellular expression.
In 1999, an Indian company, Biotechnology International Limited applied for a patent relating to recombinant mature lysostaphin, publication No. WO 01/29201, wherein the lysostaphin precursor and the signal peptide portion are deleted, the start codon ATG (encoding the first amino acid: methionine) is added directly before the recombinant mature lysostaphin gene, and the above engineered sequence is cloned into pET11b positioned behind T7 promoter, and then the lysostaphin expression is induced by IPTG. In this referenced patent application, the lysostaphin is expressed in Escherichia coli as an inclusion body, with the desired protein stored in the cell as an insoluble inclusion body, and the protein can be only isolated by disrupting the bacterial cell. During the isolation, protein denaturants has to be used, and the protein must be renatured. However, various molecules will incorrectly bind together and form into abnormal structures in the renaturation. At the same time, the cell body remnants are hard to be cleaned out in the genetically-engineered drug manufacturing process, thus negatively affecting the quality of the recombinant product.
There is no highly effective way of secretory expression of the lysostaphin in Escherichia coli. Secretory expression has the following advantages: the expressed protein exists in the culture medium as a final active form, so there is no need for renaturation of inclusion body; the desired protein is relatively easy to be isolated from the culture supernatant, with high yield; and there is less contamination of the proteins from host.